1. Field of the Invention
This invention relates to a T.sub.3 uptake immunoassay and, in particular, to a T.sub.3 uptake polarization fluoroimmunoassay.
2. Description of the Prior Art
As early as 1939, Treverrow (1) reported that hormonal iodine compounds such as thyroxine (T.sub.4) constitute the major portion of the total serum iodine. Furthermore, these iodinated organic compounds could be distinguished from serum inorganic iodide because they are bound to serum protein. Since iodine constitutes 65 percent by weight of the T.sub.4 molecule, serum protein-bound iodine (PBI) was employed as an index of serum T.sub.4. This indirect measurement of serum T.sub.4 was believed to be a good indicator of the thyrometabolic status of an individual (2,3,4). The normal range of PBI values was found to be 4-8 ug/100 ml; values below 4 ug/100 ml were consistent with hypothyroidism whereas values above 8 ug/100 ml were indicative of thyrotoxicosis (hyperthyroidism).
A major pitfall of the PBI test is its inherent lack of specificity since high levels of inorganic iodide, radio-opaque dyes and certain drugs give abnormally high values. In 1964, Murphy et al. (5) introduced their competitive protein binding analysis (CPB) for serum T.sub.4 which solved most of the nonspecificity problems associated with the PBI test. Due to the fact that CPB tests for serum T.sub.4 also required an extraction of the T.sub.4 from the remainder of the serum components, recovery variability has lead to problems both in accuracy and precision.
Recently, radioimmunoassay (RIA) has become the method of choice for measuring serum T.sub.4 (6). The RIA technique can be run directly on serum without extraction and therefore yields a simple and yet highly specific test. In general, results from RIA are from 5 to 25 percent higher than those from CPB tests.
Although the direct measurement of serum T.sub.4 is not influenced by exogenous iodine, the value obtained will be influenced by the level of the circulating T.sub.4 binding proteins. A number of states which are totally unrelated to thyroid disease may cause abnormal serum levels of T.sub.4. Changes in the serum level of circulating T.sub.4 binding proteins may cause the serum T.sub.4 level to be high or low even in the presence of normal thyroid function. Although the primary protein involved is thyroxine binding globulin (TBG), both thyroxine binding prealbumin (TBPA) and albumin also bind T.sub.4. Normally T.sub.4 is distributed as follows: 65% on TBG, 25% on TBPA, and 10% on albumin (7). In general, changes in TBG concentrations correlate much better with anomalies in thyroid function tests, such as the PBI or total T.sub.4 than do changes in TBPA (8).
Although the most accurate method to measure TBG concentrations involves the electrophoretic method of Orsorio et al. (9), the technique is too cumbersome for routine use. The method of choice which has been used for this purpose is one of the many variations of the triiodothyronine (T.sub.3) uptake test. Hamolsky et al. (10) first performed this type of test. All T.sub.3 uptake tests are designed to assess the unsaturated binding capacity of serum proteins most notably TBG. The test is based on the fact that TBG binds T.sub.3 less firmly than T.sub.4 and therefore should not upset the equilibrium set-up between T.sub.4 and TBG and, further, T.sub.3 is not normally bound to TBPA.
In the T.sub.3 uptake test an equilibrium is developed between the patient's serum, added labeled T.sub.3 and an inert exogenous binder (separating agent) of the labeled T.sub.3. One must add a sufficient amount of labeled T.sub.3 to saturate the binding sites on the TBG after which the labeled T.sub.3 that is unbound is adsorbed by the separating agent and the separating agent bound labeled T.sub.3 is counted. Therefore, when the endogenous T.sub.4 level is increased, as in hyperthyroidism, serum TBG is relatively saturated and the T.sub.3 uptake will be high. Conversely in the hypothyroid state where T.sub.4 output is low, the labeled exogenous T.sub.3 will bind to the relatively unsaturated TBG yielding a low T.sub.3 uptake.
Presently, radioassay is a method of choice for performing a T.sub.3 uptake test. However, this T.sub.3 uptake test has severe disadvantages in that radiolabeled T.sub.3, e.g., .sup.125 I-labeled T.sub.3, is relatively unstable and radioassay kits are restricted by regulations such as those governing the transport, handling, and storage of radioactive materials.
It would therefore be very desirable to develop an alternative T.sub.3 uptake test which retains the advantages present in the radioassay technique but which overcomes the two problems inherent therein, namely the relative instability of radiolabeled T.sub.3 and the restrictions placed upon radioassay kits.
Although the bases for various fluoroimmunoassays (11) such as fluorescent polarization (12-15), enhancement fluoroimmunoassay (16), fluorescent excitation transfer immunoassays (17), and fluorescent immonuabsorbent tests (18) have been described and demonstrated, to data, other than a fluorescent polarization assay of gentamicin (19) and dilantin (20) none of the above methodologies have been applied in a practical assay of proven clinical utility. For example, in the case of enhancement fluoroimmunoassays (16), it is reported that the variable levels of intrinsic serum fluorescence present an obstacle to the assay of patient samples.